Vaccines remain our most effective tool for global prevention of infectious disease. Subunit vaccines are considered safer than traditional killed or attenuated whole organism vaccines but typically lack the immunogenicity to provide long-term protection. Thus, there is an urgent need for new vaccine carriers and adjuvants that enhance the efficacy of subunit vaccines, especially those capable of directing strong cell- mediated immunity (CMI). This exploratory project will assess the utility of novel plant lectin-based antigen carriers, RTB and RTBER, to selectively orchestrate desired immune responses based on manipulation of subcellular trafficking and immune presentation of associated vaccine antigens. RTB, the non-toxic carbohydrate binding subunit B of the ricin toxin, has been shown to effectively mediate transmucosal delivery of associated vaccine antigens and elicit strong antigen-specific antibody-mediated immunity. In efforts to specifically enhance induction of CMI responses, RTB was modified to contain a C-terminal KDEL endoplasmic reticulum (ER) retrieval motif (termed RTBER). In contrast to RTB which predominately accumulates in endosomal/lysosomal compartments (the site of MHC II antigen processing) following endocytosis, RTBER should redirect associated antigen 'payload' to the ER/cytosol interface (the site of MHC I antigen processing for CMI). Both RTB and RTBER facilitate active uptake of associated antigens across mucosal surfaces and into immune responsive cells. This project tests the hypothesis that using RTBER as the vaccine antigen carrier will selectively mobilize the antigen through the retrograde ER pathway for efficient antigen processing and presentation to CD8+ T-cells via the MHC I pathway resulting in strong cell-mediated immunity. The influenza A nucleoprotein (NP), a potential universal antigen for influenza A requiring strong CMI for protection, will be used as the model antigen and the impact of RTB versus RTBER in selectively orchestrating NP-specific immune responses will be determined. The project will encompass 1) production of recombinant NP:RTB fusion proteins in a facile plant-based bioproduction system, 2) purification and characterization of the recombinant products, and 3) assessment of elicited immune responses in mice following intranasal administration of control, NP:RTB, and NP:RTBER immunogen formulations. Demonstrating RTBER efficacy in mediating strong NP-specific CMI responses is a key prerequisite to future multi-strain influenza challenge/disease protection trials. This research, if successful, will potentially identify a novel strategy for directing strong cell mediated immunity of mucosally delivered subunit vaccines and provide the foundation for follow-on experiments focused on development of universal vaccines for influenza A. PUBLIC HEALTH RELEVANCE: Vaccines represent one of the most effective approaches for protection against infectious disease agents. The proposed exploratory project will test the potential of the RTB plant lectin (the non-toxic carbohydrate binding subunit B of ricin) to facilitate both the delivery of associated vaccine antigens into immune responsive cells and to differentially present vaccine antigens to specific arms of the immune system. If successful, this research could lead to new approaches for 'needle-free' vaccine delivery and efficacy that enhance adaptive cell-mediated immunity, considered critical for protection against many viral disease agents and for the development of broadly effective universal flu vaccines.